Effect of p19 ^INK4D knockdown on MK differentiation. CD34⁺ cells were transduced with a control lentivirus or a lentivirus encoding either shRNA p19 (I) or shRNA p19 (II) GFP positive (GFP⁺). (A) p19^INK4D mRNAs were measured by QRT-PCR in GFP-positive cells at day 5 of culture. Error bars are standard deviations of the mean of 3 repeated experiments. (B) p19^INK4D protein level was analyzed at day 5 and day 9 of culture by Western blot. HDAC-1 was used as internal control of a quantity. The data illustrate 1 of 2 representative experiments that produced similar results. (C) Flow cytometric analysis of mature megakaryocytes expressing a high level of CD41 and CD42 antigens in GFP⁺ cells at day 9 of culture. (D) Real-time RT-PCR was used to quantify PF4, NF-E2, GATA-1, GPIIb, GPIIIa, FLI-1, p19^INK4D, p21, p27, cyclin D3 (CCND3), and TAL-1 mRNAs in GFP⁺ cells (at day 9 of culture). The relative expression of all these genes was calculated in comparison with HPRT mRNA. (E) GFP⁺ cells were sorted at day 9 either on the low coexpression of CD41 and CD42 (CD41⁺ CD42⁺ population) or on a high expression of both antigens (CD41⁺⁺ CD42⁺⁺ population). Cells were seeded at 2 × 10³ cells/well in 96-well plate. At day 12, the percentage of MKs forming proplatelets was estimated by counting MKs exhibiting one or more cytoplasmic processes with areas of constriction among 500 cells per well (in 5 wells). Panel C shows 1 representative analysis of 4 repeated experiments with similar results. Error bars in histograms in panels D,E represent the standard deviation of 3 repeated experiments each performed in triplicate wells.